Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: Immunological relationship with hnRNP P

Abstract

Serum from a patient showing symptoms related to autoimmunity was found to contain autoantibodies to the nuclear mitotic apparatus (NuMA) protein and to several novel nuclear antigens with estimated molecular weights of 40, 43, 72, 74 and 82 kDa. Using this serum for screening a human cDNA expression library a 2.5 kb cDNA clone was isolated which encoded the complete sequence of a protein of 633 amino acids. Sequence analysis revealed a modular structure of the protein: an acidic N-terminal region of approximately 150 amino acids was followed by three adjacent consensus sequence RNA binding domains located in the central part of the protein. In the C-terminal portion a nuclear localization signal and an octapeptide (PPPRMPPP) with similarity to a major B cell epitope of the snRNP core protein B were identified. This was followed by a glycine- and arginine-rich section of approximately 120 amino acids forming another type of RNA binding motif, a RGG box. Interestingly, three copies of a tyrosine-rich decapeptide were found interspersed in the RGG box region. The major in vitro translation product of the cDNA co-migrated in SDS-PAGE with the 82 kDa polypeptide that was recognized by autoantibodies. The structural motifs as well as the immunofluorescence pattern generated by anti-82 kDa antibodies suggested that the antigen was one of the proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Subsequently the 82 kDa antigen was identified as hnRNP R protein by its presence in immunoprecipitated hnRNP complexes and co-migration of the recombinant protein with this hitherto uncharacterized hnRNP constituent in two-dimensional gel electrophoresis. The concomitant autoimmune response to a hnRNP component of the pre-mRNA processing machinery and to NuMA, a protein engaged in mitotic events and reported to be associated with mRNA splicing complexes in interphase, may indicate physical and functional association of these antigens. Support for this notion comes from observations that concomitant or coupling of autoantibody responses to proteins which are associated with each other as components of subcellular particles are often found in autoimmune diseases.

Heterogeneous Nuclear Ribonucleoprotein P2 Is an Autoantibody Target in Mice Deficient for Mer, Axl, and Tyro3 Receptor Tyrosine Kinases

“…Indeed, preliminary results indicate that TAM Ϫ/Ϫ mice also express autoantibodies to other RNP (data not shown). This is consistent with the fact that several of the hnRNP that copurify with primary transcripts synthesized by RNA polymerase II (32) are autoantigens in lupus (33,34), rheumatoid arthritis (35), and in neurodegenerative disorders (36). It is likely that anti-P2 autoantibodies arise by a related pathogenic mechanism, as TAM Ϫ/Ϫ mice mirror the human disorders and develop immune complex deposits in kidneys, joint inflammation, and neurodegeneration (18).…”

SYNCRIP, a Cytoplasmic Counterpart of Heterogeneous Nuclear Ribonucleoprotein R, Interacts with Ubiquitous Synaptotagmin Isoforms

“…2A and Fig. 4B) as well as 82-and 74-kDa polypeptides, hnRNP R itself, and its degradates, respectively (30). When the full-length cDNA was transfected into COS-7 cells, the 66-kDa protein was overexpressed, and the protein was localized in the cytoplasm with both anti-SYNCRIP-N antibody and anti SYNCRIP-Pep antibody (Fig.…”

Rhythmic Serotonin N-Acetyltransferase mRNA Degradation Is Essential for the Maintenance of Its Circadian Oscillation

“…For the construction of pcNAT-b3ЈUTR, AANAT 3ЈUTR was amplified from bovine AANAT 10-1B cDNA (accession number NM_177509) kindly provided by C. M. Craft (8) and introduced into the EcoRI-XbaI site of plasmid pcNAT, as described above. To create pFlag-hnRNP R, a full-length coding sequence of rat hnRNP R was obtained by PCR of a rat fetal brain cDNA library (Clontech), with primers based on the published sequences of human and mouse hnRNP Rs (23,44). The coding region of hnRNP R was amplified with two primers specific for hnRNP R cDNA (5Ј primer 5Ј-AAAGCTTATGGCTAATCAGGTGAA-3Ј and 3Ј primer 5Ј-AATCTAGACTACTTCCACTGTTGC-3Ј; accession number AF441128).…”